J Pharm Pharmaceut Sci (www.cspsCanada.

org) 13(4) 510 - 523, 2010

Sulfacetamide Loaded Eudragit RL100 Nanosuspension with Potential

for Ocular Delivery
Bivash Mandal, Kenneth S. Alexander, Alan T. Riga.
Industrial Pharmacy Division, Department of Pharmacy Practice, College of Pharmacy, University of Toledo, 2801 W.
Bancroft, Toledo, OH 43606, USA.
Received, August 19, 2010; Revised, October 14, 2010; Accepted, November 5, 2010; Published, November 9, 2010.

ABSTRACT Purpose. Polymeric nanosuspension was prepared from an inert polymer resin (Eudragit
RL100) with the aim of improving the availability of sulfacetamide at the intraocular level to combat bacterial
infections. Methods. Nanosuspensions were prepared by the solvent displacement method using acetone and
Pluronic F108 solution. Drug to polymer ratio was selected as formulation variable. Characterization of the
nanosupension was performed by measuring particle size, zeta potential, Fourier Transform infrared
spectroscopy (FTIR), Differential Scanning Calorimetry (DSC), Powder X-Ray Diffraction (PXRD), drug
entrapment efficiency and in vitro release. In addition, freeze drying, redispersibility and short term stability
study at room temperature and at 40C were performed. Results. Spherical, uniform particles (size range below
500 nm) with positive zeta potential were obtained. No significant chemical interactionbetween drug and
polymer were observed in the solid state characterization of the freeze dried nanosuspension (FDN). Drug
entrapment efficiency of the selected batch was increased by pH alteration and addition of polymethyl
methacrylate in the formulation. The prepared nanosuspension exhibited good stability after storage at room
temperature and at 40C. Sucrose and mannitol were used as cryoprotectants and exhibited good water
redispersibility of the FDN. Conclusion. The results indicate that the formulation of sulfacetamide in Eudragit
RL100 nanosuspension could be utilized as potential delivery system for treating ocular bacterial infections.
_____________________________________________________________________________________
just like eye drops and reduce discomfort caused by
application of semisolid ointments. They are patient
friendly due to less frequent application, extended
duration of retention in the extraocular portion
without blurring vision.
Sulfacetamide is a sulfonamide antibiotic used
to treat conjunctivitis (11), blepheritis (12),
trachoma (13), corneal ulcer (14) and other ocular
diseased conditions (15). They are bacteriostatic in
nature and inhibit bacterial synthesis of dihydrofolic
acid by preventing condensation of pteridine with
aminobenzoic acid through competitive inhibition
of the enzyme dihydropteroate synthetase (16). The
drug is marketed as ophthalmic solution of its
sodium salt, in a USP concentration of 10% (w/v)
under the brand name Bleph-10. The usual adult
dose for conjunctivitis is 1 to 2 drops into the
conjunctival sac every 2 to 3 hours for 7 to 10 days
(17).
_______________________________________

INTRODUCTION
An exciting challenge for developing suitable drug
delivery systems targeted for ocular diseases is one
of the major focuses of pharmaceutical scientists.
There are several new ophthalmic drug delivery
systems under investigation such as: hydrogels (1);
microparticles (2); nanoparticles (3); liposomes (4);
collagen shields (5); ocular inserts/discs (6);
dendrimers (7); and transcorneal iontophoresis (8).
Nanoparticles have been found to be the most
promising of all the formulations developed over
the past 25 years of intense research in ocular
therapeutics due to their sustained release and
prolonged therapeutic benefit.
Polymeric
nanoparticles are also able to target diseases in the
posterior segment of the eye such as age-related
macular degeneration, cytomegalovirus retinitis,
diabetic retinopathy, posterior uveitis and retinitis
pigmentosa (9). Nanoparticles are solid, submicron,
colloidal particles ranging in size from 10 to 1000
nm, in which drug can be dissolved, entrapped,
adsorbed or covalently attached (10). These
colloidal particles can be applied in the liquid form

Corresponding Author: Bivash Mandal, Industrial

Pharmacy Division, Department of Pharmacy Practice,
College of Pharmacy, University of Toledo, 2801 W.
Bancroft, USA. Email: bm2306@gmail.com.

510

J Pharm Pharmaceut Sci (www.cspsCanada.org) 13(4) 510 - 523, 2010

The drug has an ionization constant of 5.4 and an

elimination half-life of 7 to 13 hours (17).
Polymeric nanosuspensions, prepared from
Eudragit RL100 and RS100, have been investigated
extensively for the ocular delivery of ibuprofen
(18), flurbiprofen (19), cloriocromene (20),
piroxicam (21), methyl prednisolone (22), and
amphotericin B (23).They are cationic copolymers
of methacrylate with 4-12% quaternary ammonium
groups. They are inert polymer resins, insoluble at
physiologic pH but have swelling properties. Due to
their capability to form nanodispersions with
smaller particle size, positive surface charge, good
stability, absence of any irritant effect on the
cornea, iris, and conjunctiva, Eudragit nanoparticles
appear to be a suitable inert carrier for ophthalmic
drug delivery.
The simplest method to prepare drug loaded
nanoparticles is the solvent displacement method
also known as nanoprecipitation method, developed
by Fessi et al. (24).The method is based on the
interfacial deposition of a polymer following
displacement of a semi-polar solvent miscible with
water. The technique is easy, less complex, less
energy consuming as well as widely applicable
without any additives for the manufacturing of
defined nanospheres (25). However, entrapment of
hydrophilic drug substances is very difficult in this
method. Ideally, nanoparticles with high drug
entrapment efficiency would reduce the quantity of
carrier required for the administration of a sufficient
amount of drug at the target site, as well as drug
wastage during manufacture. There are several
methods already reported in the literature to
improve drug entrapment efficiency of the
nanoprecipitaion method. These include: changing
the pH of the inner/external phase; addition of
excipients (fatty acids, oligomers); replacing the
salt form of the drug with the base form; and the
addition of salt to the aqueous phase (26).
Currently, no attempt has been made to
encapsulate sulfacetamide inside a polymeric
nanoparticulate carrier which could facilitate the
drug delivery to the ocular surface. Sensoy et al.
(27) have recently reported the treatment of ocular
keratis in rabbit eye using bioadhesive
sulfacetamide sodium microspheres consisting of
pectin, polycarbophil and hydroxypropylmethyl
cellulose. The rabbit eyes treated with microspheres
demonstrated significant decrease in the number of
viable bacteria in infection models when compared

to sulfacetamide alone. In another study, it was

reported that uptake of polymeric particles into
primary cultured rabbit conjunctival epithelial cells
(RCEC) was dependent on particle size (28).
Interestingly, the uptake of polymeric nanoparticles
into RECE was found to be significantly higher
compared to microparticles after topical application
to the albino rabbit eye.
Therefore, an attempt was made to prepare and
characterize sulfacetamide loaded Eudragit RL 100
nanosuspensions intended for the treatment of
ocular infections. Nanosuspensions were prepared
by the solvent displacement method using acetone
and 1 % (w/v) Pluronic F108 solution.
Physicochemical
characterization
of
the
nanosuspension was performed by measuring
particle size, zeta potential, drug entrapment
efficiency and in vitro drug release. Solid state
characterization of the freeze dried nanosuspension
was performed by Fourier Transform Infrared
spectroscopy (FTIR), Differential Scanning
Calorimetry (DSC) and Powder X-Ray Diffraction
analysis (PXRD). These techniques allow
understanding the thermal behavior, drug
crystallinity and possible occurrence of drug
polymer interaction for the freeze dried
nanosuspension. The effect of changing polymer
content, pH of the external media and addition of
polymethyl methacrylate (PMMA) on drug
entrapment efficiency was studied for the selected
batch. Freeze drying and redispersibility of the
lyophilized samples were performed for the selected
batch. Short term stability for 1 month for the
selectedbatch was also carried at room temperature
(200C) and at 40C.
MATERIALS AND METHODS
Materials
Eudragit RL 100 was purchased from Rhm GmbH
& Co. KG, Germany. Sulfacetamide (purity of 99 to
100.5%) was supplied by Spectrum Chemical Mfg.
Corp, CA. Pluronic F 108 was purchased
fromBASF Wyandotte Corp, NJ. All other
chemicals were of reagent grade. Eudragit RL 100
and Pluronic F 108 were used as supplied by the
manufacturers.
Preparation of Nanosuspension
The Eudragit RL 100 nanosuspensions were
prepared by the solvent displacement method

511

J Pharm Pharmaceut Sci (www.cspsCanada.org) 13(4) 510 - 523, 2010

similar to that employed by Fessi et al. (24). Four

different weight ratios of drug and polymer, namely
10:100 (batch B1), 20:100 (batch B2), 30:100
(batch B3) and 40:100 (batch B4) were used as
shown in Table I. Briefly, a 100 mg portion of
Eudragit RL100 and various proportions of drug
(10-40% by weight of the total polymer) were
dissolved in 10 mL of acetone. This organic phase
was poured drop wise into 20 mL of a 1% w/v of
Pluronic F-108 solution with moderate magnetic
stirring at room temperature. Nanoparticles were
spontaneously formed and turned the solution
slightly turbid. Then, acetone was removed by
continuous stirring for 20 hrs. The resulting particle
suspension was filtered through a 1.2 m cellulose
nitrate membrane filter in order to remove larger
particle aggregates. The prepared suspension was
centrifuged at 19,000 rpm at 150C f or 2 hours
(Sorvel RC-5B refrigerated super speed centrifuge,
rotor SS-34, 33300g, K 446). For selected batch, the
supernatant was removed and the sediment was
freeze dried for 48 hrs for further analysis.

Transmission Electron Microscopy (TEM)

TEM helps to visualize the inherent matrix of
individual particles and its shape. A drop of the
suitably diluted sample was placed onto a holey
carbon coated 400 mesh copper grid and dried in an
oven at 400Cfor 20 minutes. The images were taken
using a Hitachi Ultra-thin film evaluation system
(HD-2300A) in Phase contrast, Z contrast, Scanning
Electron (SE) modes.
Differential Scanning Calorimetry (DSC)
DSC (model 822e, Mettler Toledo, OH, USA) with
a Mettler MT50 analytical balance was used in
order to analyze the thermal behavior of different
samples. Indium (3-5 mg, 99.999% pure, onset
156.6C, heat of fusion of 107.5 J/g) was used to
calibrate the instrument. Samples (3-5 mg) were
accurately weighed into 100 l aluminum pans and
then crimped. The thermograms were recorded over
a temperature range of 10-2000C at a rate of
100C/min under nitrogen purge gas at 50 mL/min.
Mettler Toledo STARe software (version 8.10) was
used to analyze data.

Particle size analysis and zeta potential

measurement
The mean particle size for the formulations was
determined by Photon Correlation Spectroscopy
(PCS) with a Zetasizer Nano ZS-90 (Malvern
Instruments Ltd., UK) equipped with the DTS
software. The reading was carried out at a 900 angle
with respect to the incident beam. The zeta potential
was measured by a laser doppler anemometer
coupled with the same instrument. A potential of
150 mV was set in the instrument. Disposable
cuvettes of 0.75 mL capacity were used for all
measurements. All measurements were run in
triplicate.

Powder X-Ray Diffractometry (PXRD)

The crystalline state of the drug in the polymer
sample was evaluated by PXRD analysis. The Xray spectra were recorded with an XPert-PRO
multipurpose X-Ray diffractometer (PANalytical,
Tokyo, Japan) using Ni-filtered, CuK radiation
with a voltage of 45 kV, and a current of 40 mA
with a scintillation counter. The instrument was
operated in the continuous scanning speed of
4/min over a 2 range of 5 to 40. The samples
were ground using a mortar and pestle, placed into
the cavity of an aluminum sample holder and
packed smoothly using a glass slide. The results
were evaluated using the X-Pert Data collector
version 2.1 software.

Scanning Electron Microscopy (SEM)

In order to examine the particle surface morphology
and shape, SEM was used. A concentrated aqueous
suspension was spread over a slab and dried under
vacuum. The sample was shadowed in a cathodic
evaporator (also known as Sputter coater) with a
gold layer of 20 nm thick in an argon gas
environment at 45 mA current for 5 seconds.
Photographs were taken using a JSM-5200
Scanning Electron Microscope (Tokyo, Japan)
operated at 10 kV.

Fourier Transform Infrared spectroscopy

(FTIR)
The Fourier transform infrared analysis was
conducted to verify the possibility of interaction of
chemical bonds between drug and polymer. The
FTIR spectrum was performed using a PerkinElmer
-1

1600 spectrophotometer with a resolution of 2 cm .

The samples were scanned in the spectral region
-1

between 4000 and 400 cm by taking an average

of 8 scans per sample. Solid powder samples were

512

J Pharm Pharmaceut Sci (www.cspsCanada.org) 13(4) 510 - 523, 2010

oven dried at around 300C, finely crushed, mixed

with potassium bromide (1:10 ratio by weight) and
pressed at 15000 psig (using a Carver Laboratory
Press, Model C, Fred S. carver Inc., WIS 53051) to
form disc.The detector was purged carefully using
clean dry nitrogen gas to increase the signal level
and reduce moisture. For the analysis of the data,
the spectrum GX series model software was used.

Kinetics of drug release

In order to analyze the drug release mechanism, in
vitro release data were fitted into a zero-order (29),
first order (30), Higuchi (31), Hixon-Crowell cube
root law (32), and Korsmeyer-Peppas model (33).
Freeze
drying
and
redispersibility
of
nanosuspension
All of the four batches (B1, B2, B3, B4) were
freeze dried to obtain a dry powder. Additionally,
selected batch (B3) was taken to study the effect of
cryoprotectant on freeze drying as well as the
redispersibilityofthe drug loaded nanosuspension.
Two cryoprotectants were used; sucrose and
mannitol both at a 2.5% and 5% w/v concentration
level. The nanosuspension sample was divided into
four 2 mL parts and placed individually into small
glass vials. The vials were placed inside a Dewar
flask containing dry ice (i.e. solid carbon dioxide)
in order to supercool and freeze. The frozen
samples were placed inside a 600 mL Labconco
fast-freeze flask with attached adapter. The freezedrying process was carried out in the Virtis
Freezemobile model 12EL. Temperature was kept
about - 700C and the vacuum was kept at 162 mT.
After 48 hours, the lyophilized samples were
collected and stored in a desiccator for further
analysis.
Redispersibility of lyophilized products was
carried out by manual hand shaking in small glass
vial with distilled water. Visual observation was
done to investigate formation of any aggregates or
precipitates after shaking. Particle size and size
distribution after redispersion of the sample was
performed using Zeta potential/Particle sizer (model
NicompTM 380 ZLS, CA, USA).

Drug entrapment efficiency (DEE)

A 20 mL portion of the freshly prepared
nanosuspension was centrifuged at 19,000g for 2
hrs at 10-150C temperature using a Sorvel RC-5B
refrigerated super speed centrifuge with rotor SS-34
at
33300 g and K 446. The amount of
unincorporated drug was measured by taking the
absorbance of the appropriately diluted supernatant
solution at a 260 nm using single beam UV
spectrophotometer
(Genesis
10
UV,
Thermoelectron Corporation, USA) against
blank/control nanosuspension. DEE was calculated
by subtracting the amount of free drug in the
supernatant from the initial amount of drug taken.
The experiment was performed in triplicate for each
batch and the average was calculated.
In vitro drug release study
The Static Franz diffusion cell was used for
studying the in vitro release of the nanosuspension.
A cellulose acetate membrane (dialysis membrane
with a molecular weight cut off value of 12,00014,000, Spectra/por molecular porous membrane
tubing, 25 mm diameter, Spectrum Medical
Industries Inc., CA 90060) was adapted to the
terminal portion of the cylindrical donor
compartment. A 10 mL portion of the
nanosuspension containing drug, sufficient for
establishing sink conditions for the assay was
placed into the donor compartment. The receptor
compartment contained 90 mL of 0.2M phosphate
buffer solution of pH 7.4 maintained at 37C under
mild agitation using a magnetic stirrer. At specific
time intervals, aliquots of 1mL were withdrawn and
immediately restored with the same volume of fresh
phosphate buffer. The amount of drug released was
assessed by measuring the absorbance at 256 nm
using a single beam UV spectrophotometer
(Genesis 10 UV, Thermoelectron Corporation,
USA).

Short term stability study of nanosuspension

Prepared nanosuspension (batch B3) was chosen to
perform short term stability study of the
nanosuspension. Samples were stored in glass vials
for 1 month at room temperature (200C) and at 40C
in freeze. After 1 month, samples were visually
observed for any sedimentation. The particle size
and size distribution was performed using Zeta
potential/Particle sizer (model NicompTM 380 ZLS,
CA, USA).

513

J Pharm Pharmaceut Sci (www.cspsCanada.org) 13(4) 510 - 523, 2010

RESULTS

mV to + 24.1 mV (Table 1). This result is

consistent with the findings of Pignatello et al. (34).
The positive surface charge for the nanoparticles
was observed due to the presence of the quaternary
ammonium groups of Eudragit RL 100.

Particle size and size distribution

The presence of bluish opalescence indicated the
formation of colloidal nanosuspension (figure not
shown). The effect of the drug-to-polymer ratio on
the size of the nanoparticles was studied using four
different weight ratios of drug and polymer, namely
10:100 (B1), 20:100 (B2), 30:100 (B3) and 40:100
(B4), as shown in Table I. Batch B0 in which no
drug was added showed a mean particle size of
398.1 nm and mean polydispersity index (PI) of
0.414. The mean particle size for drug loaded
batches (B1 to B4) varied in the narrowrange from
112.4 nm to 140.6 nm although standard deviation
was higher for batch B1 and B4. The mean PI
values for the drug loaded formulation varied in the
range of 0.456 to 0.67. It could be inferred from the
results that there was no significant impact of the
drug-to-polymer ratio on the mean particle size of
the drug loaded nanosuspensions (p < 0.05). One
way ANOVA followed by the Tucky test showed
that batch B0 showed significant difference in
particle size as compared to drug loaded batches (p
< 0.05). A trend of increasing drug content in the
formulation with decreasing mean size of
nanoparticles was observed. This observation is in
conformity with the findings of Das et al. for
Amphotericin B loaded Eudragit RL 100
nanoparticles (23). The probable spatial interaction
(due to electrostatic charges) between drug and
polymer forming more compacted structure at
higher drug concentrations have resulted in
decreasing particle size. The phenomenon may be
related to viscosity.

SEM and TEM

Nanoparticle surface morphology and shape were
visualized using SEM and TEM. SEM revealed that
the drug loaded nanoparticles were found to be
distinct, spherical with a smooth surface
(Figure1A). TEM images were also in conformity
with the SEM and dynamic light scattering data for
particle size. All particles were found to be
spherical with a smooth surface for the various
batches (Figure 1B and1C). Magnification of a
single particle showed the internal cage like
structure in which the drug molecules are dispersed
uniformly throughout the polymer matrix (Figure
1D).
DSC
From the overlay of the DSC thermograms, it has
been observed that sulfacetamide is crystalline in
nature (Figure 2). It exhibited a sharp melting
endotherm at an onset temperature of 180.10C, a
peak temperature of 182.310C and a heat of fusion
of 119.7 J/gm. The drug recrystallized at an onset
temperature of 241.760C, a peak temperature of
245.090C and had energy of activation of about
80.16 J/gm. Eudragit RL 100 polymer exists as a
completely amorphous form with a glass transition
temperature (Tg) of about 600C (35). The
amorphous polymer did not show any fusion peak
or phase transition, apart from a broad signal
around 55600C due to a partial loss of residual
humidity. The thermal behavior of the freeze dried
nanoparticles suggested that the polymer inhibited
the melting of the drug crystals.

Zeta potential
The zeta potential remained in the range of positive
values for all batches and varied between + 9.16

Table 1. Mean size, Polydispersity index and zeta potential of blank and Sulfacetamide-loaded Eudragit
RL100 Nanosuspensions ( is Standard deviation, n=3)
Batch
B0
B1
B2
B3
B4

Drug to
Polymer ratio
(by wt)
0:100
10:100
20:100
30:100
40:100

Mean
size (Z average)
(nm)
398.1 21.84
140.6 49.94
127.9 28.82
118.9 8.17
112.4 40.25

514

Polydispersity
Index
0.4140.095
0.4560.075
0.5010.145
0.670.162
0.4670.137

Zeta potential

(mV)
13.030.32
18.770.45
24.11.58
9.160.43
16.470.29

J Pharm Pharmaceut Sci (www.cspsCanada.org) 13(4) 510 - 523, 2010

Figure 1: (A) SEM image of Sulfacetamide loaded Eudragit RL 100 nanosuspension (batch B3) taken at 40,000
magnification and acceleration voltage of 10 kv, (B) TEM image of Blank Eudragit RL 100 nanosuspension taken at Z
contrast mode, (C) Sulfacetamide loaded Eudragit RL 100 nanosuspension (batch B3) taken at Scanning Electron mode,
(D) TEM image of a single nanoparticle (batch B3) showing internal structure and dispersed drug molecules in the polymer
matrix.

175.050C. The most probable reason for the

appearance of slightly shifted broad endothermic
and exothermic peaks is due to melting and
crystallization of the adsorbed poloxamer present
on the nanoparticle surface.

However, the physical mixture of drug/polymer

(1:1) did not show any drug melting peak or
crystallization peak. Freeze dried drug loaded
nanosupension (batch B3) showed a broad
endothermic transition at an onset of 21.570C, a
peak at 50.890C. Similar observation was noted for
other three batches (not shown in thermograms).
This observation can be explained from the effect of
adsorbed poloxamer as surfactant onto the drug
loaded nanoparticles. Pluronic F108 exhibited a
melting onset of 55.520C, a peak of 58.510C. The
exothermic crystallization peak of Pluronic F108
was observed at an onset of 169.860C and a peak of

PXRD
In order to investigate the physical nature of the
encapsulated drug, the Powder X-ray Diffraction
technique was used. Solid state analysis of the
nanosuspension system after freeze drying showed
the probability of the drug to disperse in the
polymeric matrices as microcrystalline (36) or

515

J Pharm Pharmaceut Sci (www.cspsCanada.org) 13(4) 510 - 523, 2010

could be in semicrystalline form (37). According to

Boyer, the semicrystalline system is characterized
by amorphous and crystalline phases that are
closely associated, leading to the establishment of a
three-dimensional interphase associated with the
paracrystalline phase (crystalline phase with low
degree of organization) and a constrained
amorphous phase (38).While the polymer is
completely amorphous in nature, entrapment of
crystalline sulfacetamide (sharp intense peaks as
seen in Figure 3) into the polymeric nanoparticles
reduced its crystallinity to a greater extent. A
similar observation was noted for the other three
batches. This is evident from the disappearance of
most peaks in the nanoparticles compared to the
drug or the physical mixture of drug/polymer (1;1).
Thus, it can be inferred that the drug is present
inside the nanoparticles in a semicrystalline or
microcrystalline form. This finding was also in
agreement with the flurbiprofen loaded acrylate
polymer nanosuspension prepared by Pignatello et
al. (34).

DEE
DEE for the sulfacetamide loaded nanosuspension
was found to be in the range of 28.26 % to 35.74%
for the four batches. The low DEE values indicate
relatively low affinity of the drug with the polymer
matrix. Another explanation for poor entrapment is
probably related to the solubility and ionization of
the drug (26).
Three strategies were used to enhance DEE for
the batch B3 and included the effect of changing
polymer content, changing external phase pH and
the addition of Polymethyl methacrylate (PMMA)
in the formulation (29). Changing the content of
polymer in the formulation B3 did not improve the
DEE of nanosuspension (data not shown). When the
pH of the aqueous phase was adjusted to 3.4,
significant improvement in DEE (~ 50%) was
observed. This finding may be due to the
suppression of ionization and decrease in solubility
of drug during the formation of nanodroplets in
solvent displacement method (26). Thus, drug
molecules did not escape from the particles when
the external aqueous surfactant solution phase was
adjusted to acidic pH (3.4) which is two units below
the pKa (5.4) of the drug. When, 30 parts of PMMA
was incorporated in B3, DEE increased to about
50%.

FTIR
Pure sulfacetamide has characteristic IR peaks at
3471.93 cm-1 (NH stretch), 1686.3 cm-1 (CO), 1642
cm-1, 1596.18 cm-1, 1505.61 cm-1, 1440.51 cm-1,
1375.01 cm-1, 1322.8 cm-1 (sym SO2), 1233 cm-1,
1155 cm-1 (asym SO2). The peaks were in
conformity with the findings of Nagendrappa (39).
Figure 4 showed that the characteristic bands of the
ester groups at 1,150 - 1,190 cm-1 and 1,240- 1,270
cm-1, as well as the C = O ester vibration at 1,730
cm-1. In addition, CHX vibrations can be discerned
at 1385 cm-1, 1450 cm-1, 1475 cm-1 and 2,950 3,000 cm-1. Eudragit has characteristics IR
absorption frequency at 3437.91cm-1 (OH stretch),
2952.37cm-1 (sp3 CH stretch), 1733.89cm-1 (CO
stretch). The observed peaks were comparable with
the findings of Basu et al. (40). Freeze dried solid
sample of sulfacetamide loaded nanosuspension
(batch B3) exhibited mainly the Eudragit absorption
peaks with few overlapping peaks from the
sulfacetamide. Slight broadening of few peaks near
3500cm-1 for the physical mixture of drug/polymer
(1:1) was observed. It can be concluded that no
strong chemical interaction occurred between drug
and polymer inside the nanoparticles. Similar
observation was noted for other three batches of
drug loaded nanosuspensions.

In vitro drug release

In vitro drug release from the nanosuspension in
phosphate buffer pH 7.4 was performed by the
dialysis experiment using the static Franz diffusion
cell. The in vitro drug release profiles obtained
from the dialysis experiment was shown in Figure
5. The amount of drug incorporation in the
formulation and drug entrapment efficiency has a
direct effect on the drug release profile from the
four batches. As the content of the drug in the
formulation increased, the release rate also
increased. Batch B4 had the lowest drug entrapment
efficiency (DEE) of 28.26% with a smaller average
particle size (112.4 nm) and gave 100% drug
release within 2 hours. Batch B1 had a DEE of
31.35 % with a larger average particle size (140.6
nm) andexhibited a prolonged drug release profile
with only about 54.22% drug release after 3 hours.
A similar tendency was observed for Batch B2
(DEE 32.24% and particle size 127.9 nm) which
released about 60.46% of the drug after 3 hours.
Batch B3 with a particle size of 118.9 nm and DEE
of 35.74% showed 91.17% drug release after 3 hrs.

516

J Pharm Pharmaceut Sci (www.cspsCanada.org) 13(4) 510 - 523, 2010

Figure 2: DSC thermograms of EudragitRL100 (A), physical mixture of Sulfacetamide/Eudragit RL100 at 1:1 ratio (B),
sulfacetamide (C), Freeze dried nanosuspension batch B3 (D), Pluronic F108 (E).

Kinetics of drug release

The release data were fitted to various kinetic
models in order to calculate the release constant and
regression coefficients (R2) as seen in Table II.
Among the models tested, the drug release profiles
for batch B1 and B2 were best fitted with the
Hixon-Crowell cube root model based on the
regression coefficients of R2 of 0.97 and 0.95,
respectively. Batches B3 and B4 followed a zero
order model with a R2 of 0.98 and 0.99,
respectively. Using the Korsemeyer-Peppas
equation, which plots the logarithm of cumulative
percentage of drug release up to 60% versus the
logarithm of time, showed an excellent fit for the
model (R2~ 0.97). The diffusion exponent (n)
values for all batches were within 0.4.

Freeze
drying
and
redispersibility
of
nanosuspension
Batch B3 (drug to polymer ratio of 30/100) was
selected for freeze drying since it had the highest
drug entrapment efficiency with a small particle
size and sustained release behavior. The effect of
using cryoprotectants on redispersibility in distilled
water was investigated visually to observe the
formation of any aggregates upon manual hand
shaking. Freeze dried nanoparticles without
cryoprotectants appeared as off-white fluffy and
sheet-like materials. Using sucrose as the
cryoprotectant resulted in the formation of a white,
brittle, crystalline material with perforated
structure. Mannitol formed a white spongy, cottonlike material upon lyophilization. The freeze dried
sample without cryoprotectants did not redisperse in
water after manual hand shaking. Large aggregates
were observed.

517

J Pharm Pharmaceut Sci (www.cspsCanada.org) 13(4) 510 - 523, 2010

Figure 3: PXRD of Sulfacetamide (A), Eudragit RL100 (B), physical mixture of Sulfacetamide/ Eudragit RL100 at 1:1
ratio (C), freeze dried nanosuspension batch B3 (D)

room temperature and 40C, respectively. The

particle size for the batch B3 was 118.9 8.17 nm
before performing the stability study. It can be
inferred from the observed data that the prepared
nanosuspension B3 was stable after 1 month of
storage at room temperature and 40C.

Mannitol containing samples showed good

redispersibility upon manual shaking. No difference
was observed for 2.5% and 5% mannitol containing
samples. Sucrose containing samples showed
excellent redispersibility within a few minutes of
shaking for samples with 5% sucrose. Samples
containing 2.5% sucrose formed slight turbidity and
foaming upon shaking.
Particle size of the 5% sucrose containing batch
was 304.7 30.4 nm whereas the 5% mannitol
containing batch contained 156.2 18.1 nm
average particle size. Therefore, the 5% mannitol
containing batch appeared to be the most suitable
cryoprotectant for batch B3.

DISCUSSION
Eudragit
RL100
Nanosuspensions
were
successfully prepared by the solvent displacement
technique. In this process, nanoparticles were
spontaneously formed when the organic phase
(acetone) containing Eudragit RL 100 with/without
sulfacetamide was added dropwise into stirred
aqueous surfactant solution (1% Pluronic F 109),
resulting in a transparent solution with a bluish
opalescence. Instantaneous formation of a colloidal
suspension occurred as a result of the polymer
deposition on the interface between the organic
phase and water when partially water miscible
organic solvent (acetone) diffused out quickly into
the aqueous phase from each transient particle
intermediate.

Short term stability study of nanosuspension

The physical appearance of the B3 nanosuspension
did not change when samples were stored at 40C for
1 month. A loose, thin layer of sediment was
observed when the nanosuspension was stored at
room temperature for 1 month. However, the
sediment disappeared with slight hand shaking. The
average particle diameters were 125.2 25.1 nm
and 98.2 21.3 nm when samples were stored at

518

J Pharm Pharmaceut Sci (www.cspsCanada.org) 13(4) 510 - 523, 2010

Figure 4: FTIR spectra of EudragitRL100 (A), Sulfacetamide (B), physical mixture of Sulfacetamide/ Eudragit RL100 at
1:1 ratio (C), freeze dried nanosuspension batch B3 (D).

method (44). In this study, all batches of the

nanosuspension exhibited size distribution range
which was below 500 nm, therefore suitable for
ocular application. From the formulation point of
view, incorporation of the drug above 40% in the
formulation resulted in aggregation and separation
of the particles to form immediate white sediment.
Therefore, the study was carried out in the range of
10-40%
drug
incorporation
in
the
formulation.Electron microscopy (SEM, TEM)
revealed that particles are smooth, regular and
spherical in nature. Additionally drug particles were
found to be dispersed inside the particles when
TEM was performed.
The positive surface charge for the
nanoparticles could allow for a longer residence
time for the particles by ionic interaction with the
negatively charged sialic acid residues present in
the mucous of the cornea and conjunctiva (45).

According to the Marangoni effect, the transient

particle intermediate causes a size reduction to the
nano range (41). Formation of a colloidal
nanodispersion can be visualized by the bluish
opalescence.
This phenomenon is known as the Tyndall
effect which results from scattering of light caused
by the dispersed colloidal particles (42).
The particle size and size distributions are
critical parameters for ocular delivery purposes in
order to avoid irritation to the ocular surfaces.
Particle size for ophthalmic application should not
exceed 10 m (43). The United States
Pharmacopoeia (USP) specifies that ophthalmic
solutions should contain no more than 50 particles
with a diameter more than 10 m, 5 particles with a
diameter of not greater than 25 m, and 2 particles
with a diameter of not greater than 50 m per mL of
solution when using the microscopic particle count

519

J Pharm Pharmaceut Sci (www.cspsCanada.org) 13(4) 510 - 523, 2010

Figure 5: In vitro release of Sulfacetamide loaded nanosuspensions in phosphate buffer pH 7.4 at 370C (n=3).

Table 2. Kinetic release rate constants, correlation coefficient and diffusion exponent of various models (n=3)
Batch
Zero order
First order
Higuchi model
Hixon-crowell
Korsemeyer peppas
K0
R2
K1
R2
Kh
R2
KH
R2
K
n
R2
B1
17.876
0.915 0.784
0.868
70.587
0.892 0.630
0.975 0.057 1.953 0.986
B2
20.228
0.948 0.747
0.894
54.035
0.845 0.645
0.953 0.080 1.856 0.995
B3
30.942
0.982 0.498
0.836
34.213
0.765 0.745
0.879 0.159 2.28
0.921
B4
50.036
0.998 0.122
0.750
29.745
0.715 1.036
0.792 0.502 1.14
0.999

constancy of zeta potential with slight variation

indicates that sulfacetamide was encapsulated
within the nanoparticles and a major part of the
drug is not present on the nanoparticle surface.
Solid state characterization of FDN was
performed by DSC, PXRD and FTIR techniques.
These techniques allow us to confirm the possibility
of any significant or insignificant chemical or
physical interaction among drug, polymer,

Sulfacetamide belongs to a class of secondary

sulfonamides in which the hydrogen on the nitrogen
atom is acidic. Thus, in basic medium, the nitrogen
acquires a negative charge on the conjugate base
stabilized by resonance. The adsorbed surfactant
(Pluronic F108) present on the nanoparticles surface
may shield the particle surface, thus covering it
with the electrically neutral layers and causing a
slight shift in the surface charge (46). The relative

520

J Pharm Pharmaceut Sci (www.cspsCanada.org) 13(4) 510 - 523, 2010

surfactants or other additives of the formulation. No

significant chemical interaction was observed
among sulfacetamide, Eudragit and Pluronic.
Therefore, the inactive formulation ingredients were
compatible with the drug.
The indirect method was used to determine
DEE as described by Hou et al. (47). After
preparing the fresh nanosuspension, it was
centrifuged and the free drug present in the
supernatant was analyzed by UV-Visible
spectrophotometer using a calibration curve.
Subtracting this value from the initial amount of
drug, DEE was calculated. The method is suitable
for determining entrapment efficiency of
nanosuspension when fairly high concentrations of
free drug are present in the supernatant after
centrifugation (48). Sulfacetamide is slightly
soluble in water and has an ionization constant of
5.4. The aqueous 1% Pluronic (surfactant) solution
has a pH of about 6. Therefore, when the organic
phase is added dropwise into the aqueous surfactant
solution, part of the drug is ionized and escapes
from the nanoparticles during diffusion of the
acetone into the aqueous phase. Increasing the drug
content in the formulation increased the DEE inside
the nanoparticles. However, when the drug content
is 40% in the formulation (batch B4), saturation of
the polymer particles occurs with such a high drug
loads. The excess drug escapes from the acetone
phase into the water. Therefore, DEE dropped in
batch B4. Another possibility for the decreased
DEE at high drug content in the formulation can be
explained by possible saturation of the cationic sites
on the Eudragit by anionic drug molecules (49).
Therefore, excess drug is being lost from the
particles during its formation process.
Drug content and average particle size has
impact on drug release profile of nanosuspensions.
As the content of the drug in the formulation
increased, the release rate also increased. Batch
with the lowest drug entrapment efficiency (DEE)
and smaller average particle size gave faster drug
release. The progressive saturation of the
quaternary groups in the polymer by drug
molecules (occurred at high drug content) increased
drug release from the formulation (23). On the other
hand, batch with a larger average particle size
exhibited a prolonged drug release profile. A
correlation between drug release from the
nanosuspensions with mean particle size was
observed. Thus, it can be inferred that larger

particles have a small initial burst release and a

longer sustained release than smaller particles (50).
Among the models tested, the drug release
profiles were best fitted with the Hixon-Crowell
cube root model (for B1 and B2) and zero order
model (for B3 and B4) based on R2 values. The
diffusion exponent (n) values calculated from
Korsemeyer-Peppas equation for all batches were
within 0.4 which indicated that the drug release
mechanism followed pure Fickian diffusion.
Pignatello et al. (34) showed that the drug release
from Eudragit RL100 particles was complex in
nature which involves the occurrence of dissolutive
and diffusive phenomena. Overall the drug release
rate was faster which were probably due to the high
water permeability and swellability characteristics
of Eudragit RL 100 (51). The presence of a high
content of quaternary ammonium groups makes the
polymer more permeable to water.
CONCLUSION
In this study, the potential of Eudragit RL 100
nanosuspension intended for ocular delivery of
Sulfacetamide was investigated. Nanosuspension
was prepared by the solvent displacement technique
which is the easiest and most reproducible method
to prepare nanoparticles without need of any
sophisticated instruments. Due to formation of
nanosuspension with suitable particle size, positive
surface charge, good short term stability,
redispersibility and sustained release characteristics,
the delivery system appears to be promising for
treating ocular bacterial inflammation and infection.
Although we improved the drug entrapment
efficiency of the formulation to some extent by pH
alteration and additional of another excipient in the
formulation, the issue warrants further attention.
Additionally, long term stability study and in vivo
study should be performed in order to evaluate the
clinical potential of the delivery system.
ACKNOWLEDGEMENTS
The authors are thankful to Dr Pannee Burckel and
Dr Joseph Lawrence of University of Toledo, Ohio
for their help with instrumentation. Special thanks
to Mr Buddhadev Layek, graduate student, North
Dakota State University, Fargo for his assistance
with Dynamic Light Scattering instrument.

521

J Pharm Pharmaceut Sci (www.cspsCanada.org) 13(4) 510 - 523, 2010

REFERENCES
1.

2.
3.

4.

5.
6.

7.

8.

9.

10.
11.

12.
13.
14.

15.

16. Brown GM. Inhibition by sulfonamides of the

biosynthesis of folic acid. Int J Lepr Other Mycobact
Dis, 1967;35(4):580-589.
17. Lacy CF, Armstrong LL, Goldman MP, Lance LL.
Drug Information Handbook. 12th ed, Laxi-Comp
inc: Hudson: Ohio; 2004.
18. Pignatello R, Bucolo C, Ferrara P, Maltese A, Puleo
A, Puglisi G. Eudragit RS100 nanosuspensions for
the ophthalmic controlled delivery of ibuprofen. Eur
J Pharm Sci, 2002;16(1-2):53-61.
19. Castelli F, Messina C, Sarpietro MG, Pignatello R,
Puglisi G. Flurbiprofen Release From Eudragit RS
and RL Aqueous Nanosuspensions: a Kinetic Study
by DSC and Dialysis Experiments. AAPS
PharmSciTech, 2002;3(2):article 9.
20. Pignatello R, Ricupero N, Bucolo C, Maugeri F,
Maltese A, Puglisi G. Preparation and
Characterization
of
Eudragit
Retard
Nanosuspensions for the Ocular Delivery of
Cloricromene.
AAPS
PharmSciTech,
2006;7(1):Article 27.
21. Adibkia K, Siahi MR, Nokhodchi A, Javadzedeh
AR, Barzegar-Jalali
M, Barar
J, Mohammadi
G, Omidi Y. Piroxicam nanoparticles for ocular
delivery: physicochemical characterization and
implementation in endotoxin-induced uveitis. J Drug
Target, 2007;15(6):407-16.
22. Adibkia K, Omidi Y, Siahi MR, Javadzedeh AR,
Barzegar-Jalali M, Barar J, Maleki N, Mohammadi
G, Nokhodchi A. Inhibition of Endotoxin-Induced
Uveitis
by
Methylprednisolone
Acetate
Nanosuspension in Rabbits.J Ocul Pharmacol Ther,
2007; 23(5): 421-432.
23. Das S, Sureshb PK, Desmukhc R. Design of
Eudragit RL 100 nanoparticles by nanoprecipitation
method for ocular drug delivery. Nanomedicine:
Nanotechnology, Biology and Medicine, 2010; 6
(2): 318-323.
24. Fessi H, Puisieux F, Devissaguet JP, Ammoury N,
Benita S. Nanocapsule formation by interfacial
polymer deposition following solvent displacement.
Int J Pharm, 1989; 55(1):r1-r4.
25. Hornig S, Heinze T, Becer CR, Schubert US.
Synthetic
polymeric
nanoparticles
by
nanoprecipitation. J Mater Chem,2009;19:3838
3840.
26. Peltonen L, Aitta J, Hyvnen S, Karjalainen M,
Hirvonen J. Improved Entrapment Efficiency of
Hydrophilic
Drug
Substance
During
Nanoprecipitation of Poly(l)lactide Nanoparticles.
AAPS PharmSciTech, 2004; 5(1):article 16.
27. Sensoy D, Cevher E, Sarc A, Ylmaz M., zdamar
A, Bergiadi N. Bioadhesive sulfacetamide sodium
microspheres: Evaluation of their effectiveness in
the treatment of bacterial keratitis caused by
Staphylococcus
aureus
and
Pseudomonas

Jennifer JKD, William FM. Thermoresponsive

hydrogels as a new ocular drug delivery platform to
the poterior segment of eye. Trans Am Ophthalmol
Soc, 2008;106:206-214.
Joshi A.Microparticulates for Ophthalmic Drug
Delivery. J Ocul Pharmacol,1994;10(1):29-45.
Nagarwal RC, Kant S, Singh PN, Maiti P, Pandit JK.
Polymeric nanoparticulate system: A potential
approach for ocular drug delivery. J Control
Release, 2009;136(1):2-13.
Nagarsenker MS, Londhe VY
and Nadkarni GD.
Preparation
and
evaluation
of
liposomal
formulations of tropicamide for ocular delivery. Int J
Pharm, 1999;190(1):63-71.
Yoel G, Guy K. Use of collagen shields for ocularsurface drug delivery. Expert Rev Ophthalmol,
2008;3(6): 627-633.
Bloomfield SE, Miyata T, Dunn MW, Bueser N,
Stenzel KH, Rubin AL. Soluble Gentamicin
Ophthalmic Inserts as a Drug Delivery System. Arch
Ophthalmol, 1978; 96(5):885-887.
Vandamme TF, Brobeck L. Poly(amidoamine)
dendrimers as ophthalmic vehicles for ocular
delivery of pilocarpine nitrate and tropicamide. J
Control Release, 2005; 102(1): 23-38.
Monti D, Saccomani L, Chetoni P, Burgalassi S,
Saettone MF. Effect of iontophoresis on transcorneal
permeation 'in vitro' of two -blocking agents and on
corneal hydration. Int J Pharm, 2003; 250(2):423429.
Antoine BR, Francine BC, David B, Robert G,
Florence D. Polymeric nanoparticles for drug
delivery to the posterior segment of the eye.
CHIMIA Intl J Chem, 2005;59(6):344-347.
Kreuter J, Nanoparticles in Kreuter J(ed), Colloidal
Drug Delivery Systems, Marcel Dekker, INC, New
York, NY, pp 219-342, 1994
Roth HW, Leimbeck R, Sonnenschein B, Anger CB,
Weber S. The effective antibacterial spectrum of
sulfacetamide.
Klin
Monbl
Augenheilkd,
1992;200(3):182-186.
Abboud I, Massoud WH. Effect of blephamide in
blepharitis.
Bull
Ophthalmol
Soc
Egypt,
1972;65(69):539-543.
Chhibber PR. Treatment of trachoma. Ind J Med
Res, 1964;58:3-8.
Colomina J, Esparza L, Buesa J, Mar J. Corneal
ulcer caused by Nocardia asteroides after penetrating
keratoplasty. Med Clin (Barc), 1997; 108(11):424425.
Sridhar MS, Sharma S, Reddy MK, Mruthyunjay P,
Rao GN. Clinicomicrobiological review of Nocardia
keratitis. Cornea,1998;17(1):17-22.

522

J Pharm Pharmaceut Sci (www.cspsCanada.org) 13(4) 510 - 523, 2010

28.

29.

30.

31.

32.

33.
34.

35.

36.

37.

38.

39. Nagendrappa G. An elegant example of

chemoselective reaction. Resonance, 2008; 13(10),
929-940.
40. Basu SK, Adhiyaman R. Preparation and
Characterization of Nitrendipine loaded Eudragit RL
100 Microspheres Prepared by an Emulsion-Solvent
Evaporation Method. Tropical J Pharm Res, 2008; 7
(3): 1033-1041.
41. Pignatello R, ConsoliP,Puglisi G.In vitro release
kinetics of Tolmetin from tabletted Eudragit
microparticles. J Microencapsul, 2000;17(3):373383.
42. Marangoni C. Uber die Ausbreitung der
Tropfeneiner
Flussigkeit
auf
der
Oberflacheeineranderen. Annalen der Physik und
Chemie,1871; 219:337354.
43. Mecklenburg W. The relation between the Tyndall
effect and the size of the particles of colloidal
solutions. Kolloid-Zeitschrift, 1915;16:97-103.
44. Zimmer A, Kreuter J. Microspheres and
nanoparticles used in ocular delivery systems. Adv
Drug Del Rev, 1995;16(1):61-73.
45. Marchal-Heussler L, Maincent P, Hoffman M,
Spittler J, Couvreur P. Antiglaucomatous activity of
betaxolol chlorhydrate sorbed onto different
isobutylcyanoacrylate nanoparticle preparations. Int
J Pharm, 1990;58(2):115-122.
46. Ter VR, Fromell K, Caldwell KD. Shifts in
polystyrene particle surface charge upon adsorption
of the Pluronic F108 surfactant. J Colloid Interface
Sci, 2005;288(1):124-128.
47. Hou Z,Wei H, Wang Q,Sun Q,Zhou C, Zhan C,Tang
X,Zhang Q. New Method to Prepare Mitomycin C
Loaded PLA-Nanoparticles with High Drug
Entrapment
Efficiency.
Nanoscale
Res
Lett, 2009; 4(7): 732737.
48. Xu X, Fu Y, Hu H, Duan Y, Zhang Z. Quantitative
determination of insulin entrapment efficiency in
triblock copolymeric nanoparticles by highperformance liquid chromatography. J Pharm
Biomed Anal, 2006;41(1):266-273.
49. Quinteros DA, Rigo VR, Kairuz AF, Olivera
ME, Manzo RH, AllemandiDA. Interaction between
a cationic polymethacrylate (Eudragit E100) and
anionic drugs. Eur J Pharm Sci, 2008; 33(1):72-9.
50. Hans
ML,
Lowman
AM.
Biodegradable
nanoparticles for drug delivery and targeting. Curr
Opin Solid State Mater Sci, 2002;6(4):319-327.
51. Klarslan M, Baykara T. Effects of the
permeability
characteristics
of
different
polymethacrylates
on
the
pharmaceutical
characteristics of verapamil hydrochloride-loaded
microspheres. Journal of Microencapsulation 2004,
21(2), 175-189.

aeruginosa in a rabbit model. Eur J Pharm

Biopharm, 2009;72(3):487-495.
Qaddoumi MG, Ueda H, Yang J, Davda J,
Labhasetwar V, Lee VH. The characteristics and
mechanisms of uptake of PLGA nanoparticles in
rabbit conjunctival epithelial cell layers. Pharm Res,
2004;21(4):641-648.
Govender T, Stolnik S, Garnett MC, Illum L and
Davis SS. PLGA nanoparticles prepared by
nanoprecipitation: drug loading and release studies
of a water soluble drug. J Control Release,
1999;57(2):171-185.
Wagner JG. Interpretation of percent dissolved-time
plots derived from in vitro testing of conventional
tablets
and
capsules.
J
Pharm
Sci,
1969;58(10):1253-1257.
Higuchi T. Mechanism
of
sustained
action
medication. Theoretical analysis of rate of release of
solid drugs dispersed in solid matrices. J Pharm
Sci, 1963;52:1145-1149.
Hixson AW, Crowell JH. Dependence of reaction
velocity upon surface and agitation: I-theoretical
consideration. Ind Eng Chem Res, 1931; 23:923931.
Korsmeyer RW, Gurny R, Doelker E, Buri P, Peppa
s NA. Mechanisms of solute release from porous
hydrophilic polymers.Int J Pharm, 1983;15:25-35.
Pignatello R, Bucolo C, Spedalieri G, Maltese A,
Puglisi G. Flurbiprofen-loaded acrylate polymer
nanosuspensions for ophthalmic application.
Biomaterials, 2002;23(15):3247-3255.
Cska G, Gelencsr A, Kiss D, Psztor E, Klebovich
I, Zelk R. Comparison of the fragility index of
different eudragit polymers determined by activation
enthalpies. J Therm Anal Calorim, 2007; 87 (2):
2469-473.
Shivakumar HN, Desai BG, Deshmukh G. Design
and Optimization of Diclofenac Sodium Controlled
Release Solid Dispersions by Response Surface
Methodology. Indian J Pharm Sci, 2008; 70(1): 22
30.
Boutonnet-Fagegaltier N, Menegotto J, Lamure A,
Duplaa H, Caron A, Lacabanne C, Bauer M.
Molecular mobility study of amorphous and
crystalline phases of a pharmaceutical product by
thermally stimulated current spectrometry. J Pharm
Sci, 2002; 91: 15481560.
Boyer RF. Transitions and relaxations in amorphous
and semicrystalline organic polymers and
copolymers. In: Encyclopedia of Polymer Science
and Technology; Vol II 1977, New York: John
Wiley & Sons, Inc.; pp. 745839.

523